ABSTRACT Iron homeostasis is tightly regulated to provide this critical element for growth and survival, but to prevent the toxicity of iron excess. Abnormal regulation of systemic iron balance leads to common diseases including anemia and the iron overload disorder hereditary hemochromatosis. Hepcidin is a master regulator of overall body iron balance that acts by downregulating the cell-surface expression of the iron exporter ferroportin to control iron absorption from the diet and iron release from body stores. However, the mechanisms by which iron levels are sensed by the liver to regulate hepcidin expression are not fully understood. We have previously demonstrated that 1) hemojuvelin (HJV) is a co-receptor for the bone morphogenetic protein (BMP)-SMAD signaling pathway, which is a central transcriptional regulator of hepcidin expression in the liver, 2) BMP6 is a key endogenous ligand of HJV in the regulation of hepcidin expression and systemic iron balance, 3) the BMP-SMAD signaling pathway is critical to hepcidin regulation by iron, 4) there are at least two mechanisms by which iron activates the BMP-SMAD pathway: liver iron induces liver BMP6 mRNA expression and circulating iron stimulates SMAD signaling downstream of BMP6, 5) the BMP- SMAD signaling pathway intersects with most other known hepcidin regulatory pathways including the hemochromatosis proteins HFE and TFR2 and inflammation, and 6) modulators of the BMP-SMAD pathway regulate hepcidin expression to treat hemochromatosis and anemia of chronic disease in animal models. These studies have already yielded important insights into the pathophysiology of hemochromatosis and have identified the BMP-SMAD pathway as a novel therapeutic target for iron disorders. In this competing renewal, we will address important unanswered questions about how iron is sensed by the liver to regulate hepcidin, and the key role of the BMP-SMAD pathway in this process. In Specific Aim I, we will use our novel conditional Bmp6 KO mice in specific liver cell populations to determine the cellular source of BMP6 that regulates hepcidin expression, and we will use in vitro approaches together with an in vivo bioluminescence assay in mice to elucidate the molecular mechanisms underlying iron-mediated BMP6 induction. In Specific Aim II, we will use genetic and pharmacologic approaches in vivo coupled with biochemical and cell culture studies to identify other specific functional components of the BMP-SMAD signaling cascade that are critical for hepcidin regulation and iron homeostasis. The long-term goals of this project are to understand the role of the BMP signaling pathway in regulating hepcidin expression and systemic iron balance, to gain insights into the physiology and pathophysiology of iron homeostasis in health and disease, and ultimately to develop new therapeutic strategies for treating disorders of iron metabolism.